Bottom Line:
The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity.However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity.Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

ABSTRACTMitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

Figure 5: RNA-editing activity of TbMP99/100-minus trypanosomes. (A) Precleaved insertion editing assay of mitochondrial 20S and sub-20S fractions of wild-type (WT) and TbMP99/100 double knockdown (99/100 dkd) cells. The assay monitors the gRNA-dependent insertion of 3U nucleotides. A representation of the pre-mRNA/gRNA hybrid is depicted on the right. 5′ pre-mRNA cleavage fragment (CF). black; 3′ CF, grey. The chemical identities of the 5′ and 3′ ends are as in Figure 2A. An asterisk indicates the radiolabel. Reaction products were separated in denaturing polyacrylamide gels and visualized by phosphorimaging. A schematic representation of the reaction products is given on the right. The graph shows a quantitative analysis of the editing activity. (B) Precleaved deletion editing assay of mitochondrial sub-20S and 20S fractions of WT and 99/100 dkd cells. The assay monitors the gRNA-dependent deletion of 4Us. All annotations are as in (A).

Mentions:
Enriched editosome preparations from all three RNAi cell lines were analyzed for their in vitro RNA-editing activity by monitoring both, U-deletion and U-insertion editing (41,42). Due to the reduced molecular mass and the altered hydrodynamic properties of the editing complexes in the kd-cell lines, we analyzed sub-20S as well as 20S editosome fractions. The two skd-cell lines (99 skd and 100 skd) showed activities identical to editosome preparations derived from uninduced cells (data not shown). By contrast, editosome preparations from 99/100 dkd cells were affected in their ability to process synthetic pre-edited mRNAs. Comparing 20S editosomes from uninduced cells with sub-20S fractions from the 99/100 dkd cell line (please note, the absence of TbMP99, TbMP100 and of ligase TbMP52 amounts to 250 kDa) revealed a drop in the insertion activity by ≥60% (Figure 5A). Similarly, deletion editing was reduced by ≥90% compared to the activity derived from uninduced cells (Figure 5B).Figure 5.

Figure 5: RNA-editing activity of TbMP99/100-minus trypanosomes. (A) Precleaved insertion editing assay of mitochondrial 20S and sub-20S fractions of wild-type (WT) and TbMP99/100 double knockdown (99/100 dkd) cells. The assay monitors the gRNA-dependent insertion of 3U nucleotides. A representation of the pre-mRNA/gRNA hybrid is depicted on the right. 5′ pre-mRNA cleavage fragment (CF). black; 3′ CF, grey. The chemical identities of the 5′ and 3′ ends are as in Figure 2A. An asterisk indicates the radiolabel. Reaction products were separated in denaturing polyacrylamide gels and visualized by phosphorimaging. A schematic representation of the reaction products is given on the right. The graph shows a quantitative analysis of the editing activity. (B) Precleaved deletion editing assay of mitochondrial sub-20S and 20S fractions of WT and 99/100 dkd cells. The assay monitors the gRNA-dependent deletion of 4Us. All annotations are as in (A).

Mentions:
Enriched editosome preparations from all three RNAi cell lines were analyzed for their in vitro RNA-editing activity by monitoring both, U-deletion and U-insertion editing (41,42). Due to the reduced molecular mass and the altered hydrodynamic properties of the editing complexes in the kd-cell lines, we analyzed sub-20S as well as 20S editosome fractions. The two skd-cell lines (99 skd and 100 skd) showed activities identical to editosome preparations derived from uninduced cells (data not shown). By contrast, editosome preparations from 99/100 dkd cells were affected in their ability to process synthetic pre-edited mRNAs. Comparing 20S editosomes from uninduced cells with sub-20S fractions from the 99/100 dkd cell line (please note, the absence of TbMP99, TbMP100 and of ligase TbMP52 amounts to 250 kDa) revealed a drop in the insertion activity by ≥60% (Figure 5A). Similarly, deletion editing was reduced by ≥90% compared to the activity derived from uninduced cells (Figure 5B).Figure 5.

Bottom Line:
The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity.However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity.Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

ABSTRACTMitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.